Research Centre for Anthropology and Health (CIAS), University of Coimbra, 3000-456 Coimbra, Portugal.
Research Centre for Anthropology and Health (CIAS), University of Coimbra, 3000-456 Coimbra, Portugal; Department of Life Sciences, University of Coimbra, 3000-456 Coimbra, Portugal.
Forensic Sci Int. 2024 Mar;356:111950. doi: 10.1016/j.forsciint.2024.111950. Epub 2024 Jan 28.
Evaluation of DNA methylation (DNAm) patterns is a promising tool for age estimation. The duplex droplet digital PCR (ddPCR) method has been recently investigated for DNAm evaluation, revealing to be a potential methodology for DNAm evaluation and molecular age estimation. In this study, we evaluated DNAm levels of CpGs located at the three age-associated genes ELOVL2, FHL2 and PDE4C using ddPCR to develop an age prediction model. Blood-derived DNA samples from 58 healthy individuals (42 women and 16 men; aged 1-93 years old) were submitted to bisulfite conversion followed by ddPCR using dual-labeled probes targeting methylated and unmethylated DNA sequences. Simple linear regression statistics revealed a strong correlation between DNAm levels and chronological age for FHL2 (R = 0.948; P = 1.472 × 10) and PDE4C (R = 0.819; P = 3.917 × 10), addressing only one CpG for each gene. For the ELOVL2 gene, evaluating five CpG sites in simultaneous, revealed a strong age correlation (R = 0.887; P = 2.099 × 10) in a simple linear regression statistics and very strong age correlation (R = 0.926; P = 2.202 × 10) when using quadratic regression statistics. The multivariable regression analysis, using methylation information captured on ELOVL2 (squared), FHL2 and PDE4C genes, revealed a very strong age correlation (R = 0.970; P = 5.356 ×10), explaining 93.7 % of age variance, displaying a mean absolute deviation (MAD) between chronological and predicted age of 4.657 years (RMSE = 6.044). We postulate that the ddPCR method should be further investigated for DNAm-based age prediction, because it is a relatively simple and an accurate method that can be routinely used in forensic laboratories for testing a few numbers of markers.
评估 DNA 甲基化（DNAm）模式是一种很有前途的年龄估计工具。双液滴数字 PCR（ddPCR）方法最近被用于 DNAm 评估的研究，被证明是一种用于 DNAm 评估和分子年龄估计的潜在方法。在这项研究中，我们使用 ddPCR 评估了位于三个与年龄相关的基因 ELOVL2、FHL2 和 PDE4C 上的 CpG 位点的 DNAm 水平，以开发年龄预测模型。从 58 名健康个体（42 名女性和 16 名男性；年龄 1-93 岁）的血液衍生 DNA 样本进行亚硫酸氢盐转化，然后使用针对甲基化和未甲基化 DNA 序列的双标记探针进行 ddPCR。简单线性回归统计显示，FHL2（R = 0.948；P = 1.472×10）和 PDE4C（R = 0.819；P = 3.917×10）的 DNAm 水平与年龄呈强相关性，每个基因仅针对一个 CpG。对于 ELOVL2 基因，同时评估五个 CpG 位点，在简单线性回归统计中显示出很强的年龄相关性（R = 0.887；P = 2.099×10），在二次回归统计中显示出非常强的年龄相关性（R = 0.926；P = 2.202×10）。使用 ELOVL2（平方）、FHL2 和 PDE4C 基因上捕获的甲基化信息进行多元回归分析，显示出很强的年龄相关性（R = 0.970；P = 5.356×10），解释了 93.7%的年龄方差，显示出与实际年龄的平均绝对偏差（MAD）为 4.657 岁（RMSE = 6.044）。我们假设 ddPCR 方法应进一步研究用于基于 DNAm 的年龄预测，因为它是一种相对简单且准确的方法，可在法医实验室中常规用于测试少量标记物。
